According to current theory, force generation in muscle fibers is related to the conformational change that occurs as myosin, while hydrolyzing ATP, changes between its weakly- and strongly-binding conformations. We have elected to study the molecular mechanism of force generation by studying things that inhibit it. One very potent inhibitor of force production is alkylation of myosin's essential sulfhydryls, Cys-707 (SH1) and Cys-697 (SH2). We examined the effect of alkylating both these sulfhydryls using either the monofunctional N-phenylmaleimide, NPM (2 moles per mole myosin subfragment-1), or the bifunctional reagent, paraphenylene dimaleimide, pPDM, (1 mole per mole subfragment-1). In solution, we found three very large effects of NPM-modification of myosin. Interestingly, the effect of NPM-modification on the binding of myosin to actin is small (less than 10-fold). The three large effects on myosin are that the binding of MgATP to myosin is reduced 20,000-fold, the binding of MgATP to actomyosin is reduced 10,000-fold, and the rate constant for cleavage of the terminal phosphate of ATP also is reduced 10,000-fold. We were able to explain all three large effects by assuming (Rosenfeld and Taylor, JBC, 1984) that initial ATP binding is followed by an isomerization whose equilibrium constant is reduced from 5 x 10+4 M-1 to 5 x 10-5 M-1 by NPM-alkylation. Inhibition of this isomerization slows phosphate cleavage to the point where active force generation is not possible. In addition to looking at SH1-SH2-modified myosin and actomyosin, we also have been looking at the EDC-tethered actomyosin preparation, both in solution and in muscle fibers. EDC is a zero length cross-linker that is able to tether myosin heads to actin, apparently without inhibiting the conformational changes that myosin normally undergoes. It is then, in principle, possible, in muscle fibers, to measure the force generated by the myosin's nucleotide-driven conformational changes. We have examined the EDC crosslinking reaction in solution in order to look for conditions most suitable for crosslinking in the fiber. We have found the EDC tethering reaction in solution to be very temperature sensitive, going, for one set of conditions, from 99% at 35 degrees C to 30% at 5 degrees. In muscle fibers, we have been able to achieve a high degree of tethering of myosin to actin with a surprizing, but potentially very useful result. Contrary to reports in the literature, EDC-tethering did not seem to activate the muscle fiber, thus making it possible to measure the force generated by the myosin conformational changes in a crossbridge head having unmodified sulfhydryls. Because of our finding that NPM-modification specifically inhibits the hydrolytic step, by comparing the force in an unmodified head to that generated by a head alkylated at the SH1 and SH2 sulfhydryls by N-phenylmaleimide, it should be possible to measure the force generated specifically by the hydrolytic step.